Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture.

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

Non-mandatory immunization and its potential impact on pertussis epidemiology. / La no obligatoriedad de la vacunación y su potencial impacto en la epidemiología de coqueluche.

In this study, we performed a quantitative analysis of the potential short-term consequences on pertussis of the draft bill on Informed Consent for Immunization proposed in Argentina in 2017, which considers a non-mandatory immunization schedule for minors. We used a mathematical model of pertussis transmission, which had been previously developed by our group. It is considered that the mere presentation of the project causes a reduction in coverage because it creates suspicion on the benefits of immunization. Assuming a 5 % annual reduction in coverage for 4 years as of 2018, in the next outbreak, severe cases in infants younger than 1 year will increase more than 100 % compared to the latest outbreak, with an estimated 101 deaths. With a 10 % annual reduction in the coverages for 4 years, the next outbreak would result in an increase of number of cases of more than 200 %, with 163 deaths.

En este trabajo, se analizan cuantitativamente las consecuencias a corto plazo que tendría sobre coqueluche la sanción del Proyecto de Ley de Consentimiento Informado en Materia de Vacunación presentado en Argentina, en 2017, el cual contempla la no obligatoriedad de la aplicación de las vacunas del Calendario Nacional a los menores de edad. Se utiliza un modelo matemático para la transmisión de pertusis, desarrollado previamente en nuestro grupo. Se considera que la sola presentación del proyecto provoca una disminución en las coberturas por generar desconfianza sobre los beneficios del programa de vacunación. Asumiendo 5 % anual de reducción de las coberturas durante 4 años a partir de 2018, en el siguiente brote, los casos graves en menores del año se incrementarían en más del 100 % respecto del último brote, y se estiman 101 fallecidos. Con una reducción del 10 % anual por 4 años, el siguiente brote superaría al previo en más del 200 %, con 163 decesos.

The symbiotic defect in a Sinorhizobium meliloti lipopolysaccharide mutant can be overcome by expression of other surface polysaccharides.

In this work we have examined the extent of functional complementation in symbiosis among different Sinorhizobium meliloti surface polysaccharides including lipopolysaccharide (LPS). We show that a symbiotic deficiency associated with an LPS defect can be reversed by appropriate expression of other surface polysaccharides such as galactoglucan (EPSII) and a particular form of capsular polysaccharide (KdoPS). It is noteworthy that, while succinoglycan EPSI and LPS cannot functionally substitute for each other, they can both be replaced by the same common set of polysaccharides (i.e., EPSII/KdoPS). The complex pattern of functional complementation in symbiosis among S. meliloti surface polysaccharides was shown to be different in Medicago truncatula compared to that previously reported for M. sativa.

Global population structure and evolution of Bordetella pertussis and their relationship with vaccination.

Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

La no obligatoriedad de la vacunación y su potencial impacto en la epidemiología de coqueluche / Non-mandatory immunization and its potential impact on pertussis epidemiology

En este trabajo, se analizan cuantitativamente las consecuencias a corto plazo que tendría sobre coqueluche la sanción del Proyecto de Ley de Consentimiento Informado en Materia de Vacunación presentado en Argentina, en 2017, el cual contempla la no obligatoriedad de la aplicación de las vacunas del Calendario Nacional a los menores de edad. Se utiliza un modelo matemático para la transmisión de pertusis, desarrollado previamente en nuestro grupo. Se considera que la sola presentación del proyecto provoca una disminución en las coberturas por generar desconfianza sobre los beneficios del programa de vacunación. Asumiendo 5 % anual de reducción de las coberturas durante 4 años a partir de 2018, en el siguiente brote, los casos graves en menores del año se incrementarían en más del 100 % respecto del último brote, y se estiman 101 fallecidos. Con una reducción del 10 % anual por 4 años, el siguiente brote superaría al previo en más del 200 %, con 163 decesos.

In this study, we performed a quantitative analysis of the potential short-term consequences on pertussis of the draft bill on Informed Consent for Immunization proposed in Argentina in 2017, which considers a non-mandatory immunization schedule for minors. We used a mathematical model of pertussis transmission, which had been previously developed by our group. It is considered that the mere presentation of the project causes a reduction in coverage because it creates suspicion on the benefits of immunization. Assuming a 5 % annual reduction in coverage for 4 years as of 2018, in the next outbreak, severe cases in infants younger than 1 year will increase more than 100 % compared to the latest outbreak, with an estimated 101 deaths. With a 10 % annual reduction in the coverages for 4 years, the next outbreak would result in an increase of number of cases of more than 200 %, with 163 deaths.

Resurgence of pertussis calls for re-evaluation of pertussis animal models.

Pertussis has recently re-emerged in well-vaccinated populations most likely due to a combination of pathogen adaptation and waning of vaccine-induced pertussis immunity. Changes in genomic content of the etiologic agent, Bordetella pertussis, observed in the postvaccination era can have a bearing on the efficacy of vaccines currently in use. Moreover, protective immune responses in vaccinees wane gradually depending on their originally induced size and breadth, and memory responses may not be as regularly boosted by circulating strains as was the case in the prevaccination era. This pertussis scenario asks for new, improved vaccines with at least a longer duration of protection. Pertussis vaccine research, development and postmarketing surveillance require re-evaluation and innovation of the currently available pertussis animal models, with emphasis on the use of circulating B. pertussis strains.

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC. Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants.

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in lpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo(2)-lipid IV(A). We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A) at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo(2)-[4'-(32)P]lipid IV(A) in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC, is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.